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Taqman or SYBR? - (Jun/01/2007 )

Hello everyone,
First of all, I would like to greet this forum. rolleyes.gif It has been very helpfull to me, because I never work with DNA methylation before.
Second, I have some questions:
1. Does anybody know what is better in the real-time pcr techonology: Taqman or SYBR?
2. How can I calculate the "index of methylation" in samples? Do I need to use a reference gene in a standard sample?
3. Do I have to prepare the calibration curve in every test?
4. The use of SYBR and melting curve is a good techinic to determinate the dna methilation status?

Regards
Anna

-brstudent-

Hi Anna, welcome to methylation studies.

QUOTE (brstudent)
1. Does anybody know what is better in the real-time pcr techonology: Taqman or SYBR?

Taqman is more expensive. Probe design can be difficult in bisulfite treated templates. SYBR green is more prone to unspecifc products. Both is possible.
QUOTE (brstudent)
2. How can I calculate the "index of methylation" in samples? Do I need to use a reference gene in a standard sample?
How do you want to measure methylation? I guess bisulfite treatment and then real-time MSP? I use this equotation:
EM= Efficiency of M primer
EU= Efficiency of U primer

Formula:

%methylation = (1-[EMCtM/(EMCtM+EUCtU])*100

I think that it is sufficient to establish the primer efficiencies at the beginning. I do a nested PCR (common first round and then MSP second round) so I decided not to use a reference gene. But there are some other opinions about that.
QUOTE (brstudent)
3. Do I have to prepare the calibration curve in every test?
No, you should do a mixing experiment for calibration. If your regression line is not fitting properly, you can even correct the formula by adding the y-axis cutting point.

QUOTE (brstudent)
4. The use of SYBR and melting curve is a good techinic to determinate the dna methilation status?

Well, actually, I don't think so. Melting curves are not working so well for bisulfite treated samples. Real-Time PCR can be sued, but has the short-coming that you only monitor the CpGs within the pirmer sequence. If you want a correct quantitative analysis, you should go for pyrosequencing, MALDI-TOF based analysis or direct cycle sequencing.

K.

-kr├╝melmonster-

Thank you very much, kr├╝mel. smile.gif
You are right. I'm thinking to use bisulfite treatment and real time MSP.
Can anyone recomend some articles about this method?
Best reagrds
Anna

-brstudent-

The majority of the time, SYBR is just fine. You have to design specific amplicons for each, but SYBR is much cheaper. Primer orders are cheap, taqman starts to add up over time.

I wouldnt use the Melting Curve to quantitatively measure methylation. Not very reliable. Bis sequancing or hplc would be easier for total quantification.

-sneth-

Thank you sneth,
As I don't have any experience with both and SYBR is cheaper, I will try this one first.
Could anybody inform where can I find a detailed protocol about this method?

Thanks
Anna

-brstudent-